Chemiluminescent microemulsions

ABSTRACT

The invention is directed to a light producing microemulsion formed in theresence of a surfactant/cosurfactant pair from an oil phase medium having at least an oxalate derivative and a fluorescer compound dissolved in it and an aqueous phase medium containing at least an oxidant dissolved in it. The light produced in such a microemulsion can be used to analyze aqueous oxidant-containing samples by comparing the amount of light produced by a microemulsion formed from a known quantity of an oxidant to a similar sample containing an unknown quantity of an oxidant. The comparison can be made by any well known photosensitive means and can be computerized.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel chemiluminescent microemulsionwhich can be used as an analytic tool for the detection of oxidants inaqueous samples. In addition, the present invention relates to ananalytic technique and analytic arrangement which uses peroxyoxalatechemiluminescence in a microemulsion to determine the approximateoxidant concentration of an aqueous sample.

2. Description of the Prior Art

The detection of the approximate concentration of pollutants orcontaminants, particularly those which generate an oxidizing agent suchas hydrogen peroxide, by chemiluminescent analysis is well known. Thedetection of peroxide has been reported using luminol chemiluminescenttechniques by G. L. Kok, et al., Envir. Sci. Technol., 12, pp.1072-1076(1978); DeChatelet, L. R., et al., J. Immunol, 129, pp.1589-1593 (1982);B. Descamps-Latscha, et al., Ann. Immunol., 133, pp.349-364 (1982); andP. De Sole, et al., Adv. Exp. Med. Biol., 141, pp.591-601 (1982). Also,lucigenin chemiluminescence analysis in micellar systems has beendescribed by W. L. Hinze et al. Anal. Chem., 56, p.2180 (1984).

However, the reported methods have several drawbacks. They are not verysensitive or efficient for the detection of oxidants, particularlyhydrogen peroxide. Another drawback is that these prior art reactionsrequire operating at a very high pH which necessitates adding largequantities of a strong base. This increases the risks of introducingcontaminants and interferents such as metal ions.

The overall process of the peroxyoxalate chemiluminescence system, firstdescribed by Edward A. Chandross, Tetrahedron Letters, No. 12, p.761,(1963) and disclosed in U.S. Pat. No. 4,053,430, consists essentially ofthe reaction of an oxalic acid ester with H₂ O₂ in the presence of afluorescer compound to generate chemiluminescence. The reaction sequencecan be described by the following three steps: (1) An oxalate derivativeis oxidized by an oxidizing agent such as H₂ O₂ to form the putativedioxetanedione intermediate, (2) this intermediate breaks down andtransfers its energy to a fluorescer also present in the system, (3) thefluorescer then emits a photon. This system is very efficient. Despitethe system's broad use for illumination, the prior art methods have notbeen used in chemical analysis because of the insolubility and labilityof the chemiluminescent reagents in aqueous medium.

G. Scott, W. R. Seitz and J. Ambrose, Anal. Chem. Acta, 115, p.221(1980) report grave difficulties with attempts to find compatiblesolvents for quantifying aqueous H₂ O₂ in a peroxyoxalatechemiluminescent flow injection system. A. G. Mohan et al., "AqueousPeroxyoxalate Chemiluminescence," AD121,396, Defense TechnicalInformation Center, Cameron Station: Alexandria, Va. 22304-6145, p.16,(1982), describe the peroxyoxalate chemiluminescent reaction in acyclohexane in water emulsion. They reported no significant improvementsin results. In additional work, they reported that better results wereobtained when a detergent was included.

In U.S. Pat. No. 4,647,532, Watanabe et al. describe a method for thedetection of hydrogen peroxide using a chemiluminescent method. Thismethod involves a complex, multistep reaction of a hydrogen peroxidecomponent in the presence of an oxidizing catalyst to convert anonfluorescer substance to a fluorescer substance and then reacting thefluorescer substance with an oxalic acid diester and hydrogen peroxideto produce light.

None of the above methods is efficient in producing chemiluminescence orfor adapting chemiluminescence to analytic techniques. The mostefficient fluorescent compounds useful in peroxyoxalatechemiluminescence, including rubrene, perylene, andbis-(phenylethynyl)anthracene, are all insoluble in water. The mostefficient oxalate derivatives, including amides and esters, have strongelectron withdrawing groups that favor hydrolysis in aqueous solution.This insolubility and lability of the oxalate derivatives andfluorescers in aqueous media or other protic solvents is a limitingfactor for the successful use of peroxyoxalate chemiluminescence as alight source or in analytic chemistry.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide a suitableenvironment for the reaction of the chemiluminescent reagents in anaqueous medium.

Another object of this invention is to promote the reaction between thehydrophilic hydrogen peroxide component and the hydrophobic oxalatederivative in an aqueous medium.

An additional object of this invention is to increase the efficiency andrate of the reaction by maximizing the amounts of the components presentand facilitate their mixing by maximizing the interfacial surface areabetween components.

Yet an additional object of this invention is to isolate the fluorescercompound from quenchers which may be present in an aqueous medium.

A further object of this invention is to maintain the oxalate derivativeand fluorescer compound together at a high concentration to maximizeefficient energy transfer from the former to the latter.

Yet a further object of this invention is to provide a macroscopicallyhomogenous, transparent microemulsion that is thermodynamically stableso as to maximize light release and detectability.

These and other objects of the invention are accomplished by forming alight producing microemulsion in the presence of asurfactant/cosurfactant pair from an oil phase medium having at least anoxalate derivative and a fluorescer compound dissolved in it and anaqueous phase medium containing at least an oxidant dissolved in it. Thelight produced in such a microemulsion can be used to analyze aqueousoxidant-containing samples by comparing the amount of light produced bya microemulsion formed from a known quantity of an oxidant to a similarsample containing an unknown quantity of an oxidant. The comparison canbe made by any well known photosensitive means and can be computerized.

DETAILED DESCRIPTION OF THE INVENTION

The present invention permits peroxyoxalate chemiluminescence to occurin aqueous media with greater sensitivity and efficiency. Themicroemulsion permits the utilization of peroxyoxalate chemiluminescencefor the semi-quantitative determination of oxidants in aqueous media.The microemulsions of this invention may be formed as oil-in-water orwater-in-oil microemulsions. By the compositions and methods of thisinvention chemiluminescence can be observed and detected when themicroemulsion mixture is exposed to a sample containing an oxidant,preferably H₂ O₂, which is produced by natural processes in watersources or is added to the water system. The methods and compositions ofthis invention permit the detection of H₂ O₂ concentrations inquantities as small as 10⁻⁹ M and has been found to be particularlyeffective as an indicator of aquatic pollutants at sea.

Microemulsions (also known as Winsor IV phases or swollen micellarsolutions) have been defined as thermodynamically stable, isotropicsolutions of surfactant, oil and water. (Danielson, I. and Lindman, B.,Colloids Surf, 3, 393 (1981)). Because of their unique properties,microemulsions are particularly useful for carrying out reactionsemploying immiscible reagents. More importantly, microemulsions maycontain up to 10% oil in water or vice versa and yet be completelystable and optically clear.

Optical clarity is of special interest in this invention because it isdesirable that the reaction mixture be transparent to the wave length oflight produced by the reaction and not to turbid so as to maximize lightrelease and detectability. This property is refered to herein as themedium being optically transparent. It is most preferred that the oilphase and all compounds dissolved in the oil phase of this invention besubstantially clear and transparent. The aqueous phase of this inventionshould also be substantially clear although some turbidity is expectedif test samples are taken from bodies of water in an exposed environmentsuch as water tanks, reservoirs, lakes, rivers, and oceans.

The compositions of this invention are those microemulsions whichconsist of substantially small, about 1000Å or less, droplets of onephase suspended indefinitely in the other phase. Oil-in-watermicroemulsions are the most preferred embodiment of this invention.

Because the reaction of the aqueous miscible and immiscible reagentstakes place at the organic solvent/aqueous interface, the microemulsionfacilitates contact of the reagents by maximizing the interfacialsurface area between the aqueous and nonpolar phases. Moreover, becausethe microemulsion is thermodynamically stable--i.e., it does notseparate into oil and water phases--it makes prolonged, vigorous orultrasonic mixing unnecessary. A great variety of microemulsions havebeen formulated, however, it is well known that only particularcomponents combined in specified proportions will form stable,transparent microemulsions of tiny droplets.

The formation of a microemulsion of this invention requires the presenceof about 10 to 30% by weight of an ionic or nonionicsurfactant/cosurfactant pair. The term surfactant/cosurfactant pair asused herein is defined as surface active agents that lower the surfacetension of a liquid or the interfacial tension between two liquids.Typical examples of suitable surfactant/cosurfactant pairs which may beemployed include sodium dodecyl sulfate/1-butanol; TritonX-100/1-hexanol; cetyltrimethylammonium bromide/1-butanol; Brij96/1-butanol; sodium oleate/1-butanol. Triton is a trademark product ofRohm and Hass and Brij is a trademark product of ICI America. Of course,any surfactant/cosurfactant pair can be used which permits the formationof an optically transparent microemulsion.

The oil phase material may be any of the water immiscible, nonpolarorganic materials which are optically clear and will form opticallyclear microemulsions. The materials may be aliphatic or aromaticsolvents. Also, the oil phase material should be non-reactive with thereagents and compounds likely to be encountered in the microemulsion,and the materials must be solvents for the fluorescer materials andoxalate derivatives. Preferred oil phase materials include toluene andtetradecane.

Any aqueous media from distilled water to sea water can function as theaqueous phase in microemulsions of this invention. Generally, lowelectrolyte concentrations are preferred with nonionic microemulsions(for example, those containing Triton X-100, Tween or Brij-96), whereashigher ionic strengths are preferred with ionic microemulsions (forexample, those containing sodium dodecyl sulfate, oleic acid, orcetyltrimethylammonium). In the preferred embodiment the aqueous phasecontains about 0.4 M NaCl in addition to the oxidant material.

According to the present invention, the reactants of thechemiluminescent system are kept separate from the microemulsion mediumuntil chemiluminescence is desired. The order of admixing the reactantsin the microemulsion is not critical. The reactants may be admixed in asingle step or in a series of steps. Preferably, the fluorescer andoxalate derivative are dissolved in the oil phase, to which are addedsuccessively the surfactant/cosurfactant mixture, and an aqueous sample.

The aqueous sample contains at least the oxidant. The aqueous sample canalso contain salts, color enhancers or catalysts such as sodiumsalicylate. Together, in the proper proportions, these materials form aclear, transparent homogenous phase. When the system is just used togenerate chemiluminescent light, hydrogen peroxide is used as theoxidant.

The molar concentrations (moles per liter of solution) of the componentsof the chemiluminescent system, described herein, may vary considerablywithout changing the ability to use the system for light generation andhence, semi-quantitative analysis. It is only necessary that thecomponents be present in sufficient concentrations to obtainchemiluminescence.

The molar concentration of the oxalate derivative is in the range of 1to 20 mM, preferably about 5 mM. The molar concentrations of thefluorescer compound used is from 10⁻⁶ M to 1 mM, preferably 0.2 mM.

The detectable molar concentration of the unknown oxidant or thestandard hydrogen peroxide is from about 10⁻⁹ M to 1 M. With improveddetection methods, it is expected that even lower concentrations ofoxidant materials should be detectable. The molar concentration of thesodium salicylate catalyst used is from 1 mM to 30 mM, preferably 10 mM.

The color of the chemiluminescent emission corresponds to the knownfluorescence of suitable fluorescers. Illustrative examples of thefluorescent compounds employed herein include the following:

1-chloro-9,10-bis (phenylethynyl) anthracene; perylene;2-chloro-9,10-bis (p-methylphenyl) anthracene, 6,13-bis(p-methylphenylethynyl) naphthacene; many fluorescent aromatic amines,including 3-aminofluoranthene, 1-anilinonaphthalene-8-sulfonate,2-toluidinylnaphthalene-6-sulfonate, aminopyrene, Dansyl amino acids,fluorescein and rhodamine derivatives; polynuclear aromatic hydrocarbonsand their derivatives, including rubrene sulfonate and pyrene; andcoumarins. Many other fluorescers are known to produce light inperoxyoxalate chemiluminescent reactions and any of these can be used inthis invention. The efficiency of each fluorescer is related to itsoxidation potential. The selection of a particular fluorescer will bewithin ordinary skill of people working in the field.

Any oxalic acid bis-amide or bis-ester with electron withdrawingsubstituents on the amine or alcohol moieties will serve in thereaction. These are referred to generically herein as oxalatederivatives. Methods of preparation for many of these oxalatederivatives, particularly acid esters, are well described in theliterature. The American Cyanamid Company has described dozens of suchcompounds. Several of the esters are available from commercial sources.Typical oxalate derivatives used in this invention include thefollowing: bis (2-(carboisopentyloxy)-3,5,6-trichlorophenyl) oxalate,bis [N-2-(N'-(N'- methyl) morpholinium)ethyl-N-trifluoromethylsulfonyl]oxamide bis-trifluoromethanesulfonate,bis (2,4,6-trichlorophenyl) oxalate, and bis (2,4-dinitrophenyl)oxalate.

Additionally, it has been found that a significant proportion of theoxalate derivative (0.2%) remains intact in the microemulsion after 16hours. Although the oxalate derivative is not stable indefinitely in themicroemulsion system, a half-life of about 1 hour is generallydisplayed.

The initial intensity approximately doubles as the temperature is raisedfrom 6° to 52° C. It should be noted, however, that the decay rate alsoincreases sixfold, which suggests that raising the temperature increasesthe reaction rate without really changing the total quantum yield.

The intensity of the light generated by the reaction is proportional tothe amount of oxidant present if all other factors, such as the amountof flourescer, are the same. The approximate quantity of an uknownoxidant can be estimated by comparing the light output of a known sampleor samples to the light output of an unknown sample.

In general, the comparison is done by providing a substantiallyoptically transparent oil phase material for a microemulsion. Dissolvingat least a fluorescer compound and an oxalate derivative in the oilphase material. Taking equal aliquots of the oil phase material to format least two oil phase material portions. It is good practice to haveseveral known standardizing samples. When that practice is followed, analiquot is made for each sample. The microemulsion requires asurfactant/cosurfactant pair. To maintain equal conditions, an aliquotfor each sample of a surfactant/cosurfactant pair is also taken. Atleast one aqueous sample containing a known quantity of an oxidantmaterial is prepared. To improve the quantitative accuracy, it ispreferred to have several known samples of different concentrations. Anaqueous sample containing an unknown quantity of an oxidant is alsoprepared. The volume of the known and unknown samples should be thesame. The known containing aqueous sample or samples, an aliquot of saidsurfactant/cosurfactant pair, and an aliquot of said oil phase materialare mixed to form a known containing microemulsion sample or samples.The amount of light produced in the known containing microemulsionsample is read with a photo-sensitive reading means such as a SpexFluorolog 2 (or other) fluorimeter operated without a lamp, aphotodiode, charge-coupled device, photo film, or a luminometer.Similarly, the unknown-containing aqueous sample, an aliquot of saidsurfactant/cosurfactant pair, and an aliquot of said oil phase materialis combined to form an unknown containing microemulsion sample and itslight output is read in the same device used for the known samples.Lastly, the readings of the known and unknown containing samples arecompared and the approximate quantity of oxidant in the unknown sampledetermined. Of course, this can be done automatically by computer.

Having described the invention the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention describedin this application.

EXAMPLE 1

The oxalate ester (bis (2-(carbopentyloxy)-3,5,6-trichlorophenyl)oxalate (1.3 mg/ml total volume) and the fluorescer compound(1-chloro-9,10-bis (phenylethynyl) anthracene (1 mg/ml) are dissolved intoluene. The surfactant/cosurfactant pair is sodium dodecylsulfate/1-butanol (3:5, w/w). Sodium salicylate catalyst (0.2 mg/ml) isadded to the aqueous sample. The sample is made saline by adding salt toa concentration of 0.4M NaCl. The standard or known is 100 ul of 30% H₂O₂.

Aliquots of the oil phase material and surfactant/cosurfactant pair aretaken and the three parts are combined. When the oil phase materialaliquot, surfactant/cosurfactant pair aliquot, and aqueous samples areeach combined an oil/water microemulsion (0.09: 0.43: 0.48, v/vrespectively) is formed. Readings are taken using a Spex Fluorolog 2fluorimeter operated without a lamp. The readings are compared and thequantity of unknown oxidant estimated. The quantum yield of the known isapproximately 2%.

EXAMPLE 2

Following the procedure of Example 1 Brij-96/1-butanol (2:1, w/w) isutilized as the surfactant/cosurfactant pair in an oil:water:c/sconfiguration (0:10: 0.40: 0.5, v/v) and water:oil:c/s configuration(0.63: 0.06: 0.31, v/v).

EXAMPLE 3

Following the procedure of Example 1 Triton X-100/1-hexanol (4:1, w/w)is utilized as the surfactant/cosurfactant pair in an oil:water:c/sconfiguration (0.49: 0.02: 0.49 v/v) and water:oil:c/s configuration(0.06: 0.47: 0.47 v/v).

EXAMPLE 4

Following the procedure of Example 1, cetyttrimethylammoniumbromide/1-butanol (1:1, w/w) is utilized as the surfactant/cosurfactantpair in an oil:water:c/s configuration (0.06: 033: 0.61, v/v). Thequantum yield of this reaction is very low and can be attributed to thepresence of the bromide ion, a well-known fluorescence quencher.

EXAMPLE 5

Following the procedure of Example 1, perylene is utilized as thefluorescent compound. The same procedure is repeated using2-chloro-9,10-bis (p-methoxyphenyl)anthracene, 6,13-bis (p-methylphenyl)ethynyl) naphthacene, and rubrene as fluorescers, respectively. Theintensity of the reaction generally increases with fluorescerconcentration but no increase in intensity over 1 mg/ml is observed at10 mg/ml concentration.

EXAMPLE 6

Following the procedure of Example 1, bis [N-2-(N'-(N'-methyl)morpholinium) ethyl-N-trifluoromethylsulfonyl] oxamidebis-trifluoromethanesulfonate is utilized as the oxalate derivative. Thesurfactant/cosurfactant pair, fluorescer and microemulsion configurationare the same as in Example 1.

EXAMPLE 7

Following the procedure of Example 3, bis (2,4,6-trichlorophenyl)oxalate is utilized as the oxalate derivative. Note that this oxalatederivative was not solubilized well in the sodium dodecylsulfate/1-butanol system. The other oxalates also seemed to be lesseffective than the commercially used bis(2-carbopentyloxy)-3,5,6-trichlorophenyl)oxalate.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

What is claimed is:
 1. A composition for producing light byperoxyoxalate chemiluminescence comprising a microemulsion which isoptically transparent to light produced by peroxyoxalatechemiluminescence, said microemulsion comprising:an oil phase containingat least a fluorescer compound and an oxalate derivative, an aqueousphase containing at least an oxidizing agent, and asurfactant/cosurfactant pair present in an amount sufficient to formsaid microemulsion.
 2. A composition according to claim 1, wherein theoxidizing agent is hydrogen peroxide.
 3. A composition according toclaim 2, wherein the oxalate derivative is an oxalic acid ester selectedfrom the group consisting of bis(2-(carboisopentyloxy)3,5,6-trichlorophenyl) oxalate, bis [N-2-(N'-(N'-methyl) morpholinium)ethyl-N-trifluoromethylsulfonyl] oxamide bis-trifluoromethanesulfonate,bis (2,4,6-trichlorophenyl) oxalate, bis (2,4-dinitrophenyl) oxalate andamides.
 4. A composition according to claim 3, wherein the oxalate acidester is added to the system in an amount of 1.3 mg/ml.
 5. A compositionaccording to claim 2, wherein the fluorescer compound is a memberselected from the group consisting of (1-chloro-9, 10-bis(phenylethynyl) anthracene, perylene, (2-chloro-9, 10-bis(p-methoxyphenyl) anthracene, (6,13-bis (p-methylphenyl) ethynyl)naphthacene, rubrene, fluorescein and rhodamine derivatives, coumarins,rubrene sulfonate, pyrene, amino pyrene, Dansyl amino acids,3-aminofluoranthene, 1-anilinonaphthalene-8-sulfonate, and2-toluidinylnaphthalene-6-sulfonate.
 6. A composition according to claim5, wherein the fluorescer compound is added to the system in an amountof 1 mg/ml.
 7. A composition according to claim 2, also containing acatalyst.
 8. A composition according to claim 7, wherein the catalyst issodium salicylate.
 9. A composition according to claim 8, wherein thesodium salicylate is present in the amount of 0.2 mg/ml.
 10. Acomposition according to claim 2, wherein the hydrogen peroxide ispresent at a concentration of no more than between about 10⁻⁶ M andabout 1M.